Many protein-based biopharmaceuticals are isolated from human plasma. The limited supply of the raw material human plasma, which relies partially on voluntary blood donation, combined with the generally low concentration, high fragility, and limited yield in purification of blood plasma proteins make the manufacturing of this class of medicaments difficult and expensive. There is thus a need to improve the efficiency of methods of purification of blood plasma proteins, so that as many medically relevant proteins as possible can be isolated from the same sample of human plasma in the highest yield achievable.
Protein separation and purification processes for human plasma proteins present unique challenges due to the variety of proteins, the varying nature of possible contaminants and/or impurities associated with each protein preparation, and the large quantity of protein usually needed for the production of biopharmaceuticals. Purification technologies generally involve a series of purification steps with the objective of isolating a single protein target.
Alpha-1-antitrypsin (AAT) and Apolipoprotein A-I (ApoA-I) are examples of human plasma proteins that can be manufactured into biopharmaceuticals. Methods to purify these proteins using dedicated purification processes have been described. For example PCT Publication No. WO04060528 describes a purification process for AAT and U.S. Pat. No. 5,089,602 describes the purification of ApoA-I, each process starting from human blood plasma fractions and each leading to a single protein product.
We have now developed methods allowing the purification of AAT and ApoA-I starting from the same fraction of human plasma. These methods are suitable for large-scale purification, thus providing the basis for industrially applicable manufacturing processes. The invention provides methods for separating AAT from ApoA-I at the initial stages of purification, so that the same starting material can be used as a starting material to purify both proteins, and methods to produce pharmaceutical-grade AAT and ApoA-I after said separation.
ApoA-I is a 28 kDa major protein constituent of high-density lipoprotein (HDL) and plays a key role in the reverse transport of cholesterol from the periphery to the liver for excretion or recycling.
ApoA-I particularly in reconstituted HDL-like particles has long been described as having therapeutic potential. Only recently a study was published which underscores this potential (JAMA (2007); vol. 297, p. 1675-1682).
A variety of purification techniques for ApoA-I have been developed.
One of the most common ways to purify ApoA1 on a small scale is to use ultracentrifugation in order to isolate HDL followed by a separation of ApoA-I from the HDL-particle. There are several different ways to purify ApoA-I from HDL, including solvent extraction. Ultracentrifugation is a very time-consuming method, and it is not suitable for large-scale isolation.
Methods using plasma as starting material that do not include ultracentrifugation have also been described, for example, chromatographic purification (Ross S. E. et al., Rapid chromatographic purification of apolipoproteins A-I and A-II from human plasma, Analytical Biochemistry 149, p. 166-168 (1985)) and purification using gel-filtration HPLC (Tricerri A. et al., A rapid and efficient procedure for the purification of human apolipoprotein A-I using gel-filtration HPLC, IJBC, 1, p. 159-166 (1994)). Other methods that use fractions from cold ethanol fractionation of human plasma as the starting material have also been published (Peitsch et al, A purification method for apolipoprotein A-I and A-II, Analytical Biochemistry, 178. p. 301-305 (1989)).
EP0329605 to Rotkreuzstiftung Zentrallaboratorium Blutspendedienst SRK and Lerch et al., Isolation and properties of apolipoprotein A for therapeutic use, Protides Biol. Fluids, 36, p. 409-416 (1989), relate to the preparation of apolipoproteins from fractions of human blood plasma containing lipoproteins. Both publications report that precipitates B and IV of a cold ethanol fractionation process can be used as starting material for producing ApoA-I. Use is made of buffers containing high ethanol concentrations, optionally with an organic solvent, for precipitating contaminants. The precipitates are solubilized in guanidine hydrochloride, which is subsequently removed by gel filtration or diafiltration. An anion-exchange chromatography step is included to bind the contaminants, while the ApoA-I passes through. Optionally it is proposed to concentrate ApoA-I by adsorption onto a second ion exchange resin.
WO9807751 also reports the use of ion-exchange chromatography for the isolation of ApoA-I.
Alpha-1-antitrypsin (AAT), a major serine endopeptidase inhibitor, is present in human plasma at a concentration of about 1.9 to 3.5 g/l. This glycoprotein of about 53 kDa is produced in the liver and inhibits neutrophil elastase, an enzyme involved in the proteolysis of connective tissue especially in the lung. AAT has three N-glycosylation sites at asparagine residues 46, 83, and 247, which are glycosylated by mixtures of complex bi- and triantennary glycans. This results in multiple AAT isoforms, having isoelectric points in the range of 4.0 to 5.0. Protease inhibition by AAT is an essential component of the regulation of tissue proteolysis, and AAT deficiency is implicated in the pathology of several diseases. Individuals who inherit an AAT deficiency, for example, have an increased risk of suffering from severe early-onset emphysema, the result of unregulated destruction of lung tissue by human leukocyte elastase. The administration of exogenous human AAT has been shown to inhibit elastase and is associated with improved survival and a reduction in the rate of decline of lung function in AAT-deficient patients (Crystal et al., Am. J. Respir. Crit. Care Med. 158:49-59 (1998); see R. Mahadeva and D. Lomas, Thorax 53:501-505 (1998) for a review.)
Because of its therapeutic utility, commercial AAT production has been the subject of considerable research. Much progress has been made in the production of recombinant AAT in E. coli (R. Bischoff et al., Biochemistry 30:3464-3472 (1991)), yeast (K. Kwon et al., J. Biotechnology 42:191-195 (1995); Bollen et al., U.S. Pat. No. 4,629,567), plants (J. Huang et al., Biotechnol. Prog. 17:126-33 (2001)), and by secretion in the milk of transgenic mammals (G. Wright et al., Biotechnology, 9:830-834 (1991); A. L. Archibald, Proc. Natl. Acad. Sci. USA, 87:5178-5182 (1990)). However, the isolation of AAT from human plasma is presently the most efficient practical method of obtaining AAT in quantity, and human plasma is the only FDA-approved source.
A number of processes for isolating and purifying AAT from human plasma fractions have been described, involving combinations of precipitation, adsorption, extraction, and chromatographic steps. Most published processes for AAT isolation begin with one or more fractions of human plasma known as the Cohn fraction IV precipitates, e.g. Cohn fraction IV, or more specifically fraction IV1, and fraction IV1-4 as well as precipitates of Kistler-Nitschmann supernatant A or A-I, which are obtained from plasma as a paste after a series of ethanol precipitations and pH adjustments (E. J. Cohn et al., J. Amer. Chem. Soc., 68:459-475 (1946); P. Kistler, H. S. Nitschmann, Vox Sang., 7:414-424 (1962)).
Glaser et al., Preparative Biochemistry, 5:333-348 (1975), describes a method for isolating AAT from Cohn fraction IV1 paste. The paste is stirred in a phosphate buffer at pH 8.5 in order to reactivate the AAT, which is largely inactivated by the pH of 5.2 employed in the Cohn fractionation. After dialysis and centrifugation, the supernatant is subjected to two rounds of anion exchange chromatography at pH 6.0 to 7.6 and at pH 8.6, followed by further chromatographic processing at pH 7.6 and at pH 8.0, to produce AAT in about a 30% overall yield.
Lebing and Chen, in U.S. Pat. No. 5,610,285, describe a purification process that employs an initial anion exchange chromatography step, followed by cation exchange chromatography at low pH and low ionic strength, to purify human AAT from plasma and plasma fractions. The cation chromatography takes advantage of the fact that active AAT does not bind to the ion exchange column under these conditions while contaminating proteins, including denatured AAT and albumin, are retained.
Coan, in U.S. Pat. No. 4,697,003, describes a method for isolating AAT from various Cohn plasma fractions comprising the removal of ethanol and salts from an AAT-containing fraction, followed by anion-exchange chromatography on DEAE cellulose or a similar material under conditions so that the AAT is retained on the column while undesired proteins are eluted. Coan also describes “pasteurization” at about 60° C. or more for about 10 hours, which is stated to be sufficient to render hepatitis viruses non-infective.
Glaser et al., in Anal. Biochem., 124:364-371 (1982) and also in European Patent Application EP 0 067 293, describes several variations on a method for isolating AAT from Cohn fraction IV1 precipitate that comprises the steps of (a) dissolving the paste in a pH 8.5 buffer, (b) filtering, (c) adding a dithiol such as DTT, and (d) precipitating denatured proteins with ammonium sulfate. Glaser et al. describe one variation in which treatment with DTT is carried out in the presence of 2.5% AEROSIL™ fumed silica, prior to precipitation with 50% saturated ammonium sulfate. Recovery of AAT was as good as it was in the absence of the silica, and the purification factor was improved by about 70%. Glaser states that Aerosil 380 may be used in the process to remove alpha- and beta-lipoproteins.
Mattes et al., in Vox Sanguinis 81:29-36 (2001), and in PCT Publication WO 98/56821, U.S. Pat. No. 6,974,792 and U.S. Patent Publication 2002/0082214, discloses a method for isolating AAT from Cohn fraction IV that involves ethanol precipitation, anion exchange chromatography, and adsorption chromatography on hydroxyapatite. The latter step is reported to remove inactive AAT, providing a product with very high specific activity, which, according to the inventors, is due to the enrichment of AAT isoforms with pl values of 4.3 and 4.4 said not to be present in other high purity AAT preparations known in the art.
Key et al. in PCT Publication WO 04060528 discloses a method for isolating AAT from AAT comprising fractions, preferentially from Cohn fraction IV1-4, suspending the AAT-containing protein mixture in a buffer under conditions that permit the AAT to be dissolved; contacting the resulting suspension with a disulfide-reducing agent to produce a reduced suspension; contacting the reduced suspension with an insoluble protein-adsorbing material; and removing insoluble materials from the suspension, further to be combined with ionic exchange chromatography and hydrophobic interaction chromatography.
Ralston and Drohan, in U.S. Pat. No. 6,093,804 describe a method involving the removal of lipoproteins from an initial protein suspension via a “lipid removal agent,” followed by removal of “inactive AAT” via elution from an anion-exchange medium with a citrate buffer. The lipid removal agent is stated to be MICRO CEL™ E or Chromosorb E™, a synthetic hydrous calcium silicate. In the presence of a non-citrate buffer, the anion-exchange medium binds active AAT while allowing “inactive AAT” to pass through. A citrate buffer is specified for subsequent elution of the AAT from the anion exchange medium, and also for later elution from a cation-exchange medium. Ralston and Drohan do not describe the use of a disulfide-reducing agent. The process is stated to provide AAT with a product purity of >90% (and >90% of the purified AAT to be active AAT) and manufacturing scale yields of >70%. Ralston and Drohan state that Cohn Fraction IV1 preparations in particular contain a significant amount of ApoA-I and point out that this has the effect of inhibiting column flow and capacity during purification. They report that treatment of the protein mixture suspension with the above mentioned “lipid removal agent” removes ApoA-I.
Bauer describes a method of AAT purification in PCT Publication WO 05027821 starting from different Cohn fractions, preferably fraction IV-1, the removal of contaminating substances (i.e., lipids, lipoproteins and other proteins), and separation of active from inactive AAT by sequential chromatography steps. Bauer does not mention that purifying ApoA-I would be desirable; to the contrary. Bauer points out that ApoA-I inhibits column flow and reduces capacity during purification and proposes to remove the contaminating ApoA-I by binding it to fumed silica (Aerosil™). Bauer neither discloses whether ApoA-I can be eluted from fumed silica nor suggests that this would be desirable.
While AAT is an effective treatment for emphysema due to alpha-1-antitrypsin deficiency, treatment is very costly (currently about $25,000 per year) due to the limited supply of protein and the complex manufacturing process. There remains a need for more efficient and cost-effective methods for isolating human AAT from plasma and other complex protein mixtures containing AAT.
In almost all purification methods for AAT discussed above, lipoproteins, including ApoA-I, are discarded as a contaminant, usually being still bound to a lipid removal agent. On the other hand, published purification methods for ApoA-I discard AAT in a mixture with many other plasma proteins.
Purification of both AAT and ApoA-I from the same fraction of human plasma, is only mentioned in the above cited U.S. Pat. No. 6,093,804, where it is stated that ApoA-I can be separated from AAT by adsorption to a synthetic hydrous calcium silicate and subsequently eluted with 0.5 N NaOH before further downstream processing. It is not disclosed how pharmaceutical purity grade AAT and ApoA-I can be obtained from a purification process starting from the same human plasma fraction. In fact, a later application from the same applicant (American Red Cross, PCT Publication WO 05027821) points out that this method is not suitable for large-scale preparation, specifically pointing out that the method as described in U.S. Pat. No. 6,093,804 is only efficient for small to mid-scale processing of source material in the range of a few kilograms.
Moreover, elution of ApoA-I with 0.5 N NaOH as proposed in U.S. Pat. No. 6,093,804 creates a high alkaline environment of about pH 13.69 that will lead to the partial or even complete deamidation of ApoA-I (see Johnson A. et al., Biochem. Biophys. 1989, 268(1): 276-86) and possibly to irreversible denaturation. As biopharmaceuticals usually loose their biological activity and worse are prone to eliciting immunogenic reactions if deamidated and/or denatured, there is a need to develop further methods of purification that cause less or no denaturation.